The project is directed toward the elucidation of some of the synthetic mechanisms involved in DNA replication. DNA replication intermediates, plasmid structure, whole chromosomes, and specific enzyme activities will be examined. A major goal is to determine the strand specificity of the two discrete size classes of small DNA intermediates generated during discontinous synthesis. A companion approach is to determine the gene products involved in the synthesis and extension of each size class. A second project is to analyze both genetically and biochemically a set of putative RNaseH mutants to determine if this enzyme plays a role in normal DNA replication. We also intend to exploit our finding that one temperature-sensitive mutant fails to make additional replication forks at the restrictive temperature but continues to use those formed at the permissive temperature. Another project is to study DNA replication in vivo and in vitro in cells containing multiple copies of a particular DNA gene. Such cells are constructed by transformation with recombinant hybrid plasmid DNA. Finally, a suppressed nonsense mutation which results in a temperature-sensitive gyrase activity will be further characterized genetically and phenotypically.